INSTITUTION:Department of Biochemistry and Biophysics, University of Hawaii School

of Medicine, Honolulu, HI 96822, U.S.A.(808)bemorton@hawaii.edu

ABSTRACT

In humans,
(-)-Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of
marijuana, causes many effects similar to those produced by benzodiazepines,
barbiturates, and alcohol, each of which act on the chloride channel regulated
by the gamma-aminobutyric acid (GABA) type A receptor.Here, using rat brain synaptoneurosomes, we
have found that THC significantly increased basal and GABA-mediated chloride-36
ion uptake in a manner which could be blocked by picrotoxin or
bicuculline.This provides evidence of
significant GABA-A receptor-complex involvement in cannabis intoxication.

Key Words:Cannabis savita; Cannabinoids; GABA A receptor;

Chloride channel complex; Chloride uptake; (Rat)

1.INTRODUCTION:

(-)-Delta-9-tetrahydrocannabinol (THC) is the major psychoactive
compound present in marijuana (Cannabis savita) (Pradham, 1984; Razdan,
1986).In human subjects, THC alters
anxiety, muscle tension, thermoregulation, memory, the sense of time, nausea,
pain, and a number of other functions (Bachman et al., 1979; Hollister,
1986).The prevalent view was that THC,
a very hydrophobic molecule, produced its CNS functional alterations by
changing general fluidity and permeability characteristics of neuronal
membranes (Hillard et al., 1985; Martin, 1986).Although reasonable, this view could not explain why compounds
with similar lipid solubilities as THC had little effect on the CNS (Kriwacki
and Makriyannis, 1989; Razdan, 1986), while such drugs benzodiazepines (BZ),
barbiturates, and muscimol, affecting specific receptors produced many of the
behavioral changes caused by THC.

In the 1980s
researchers began to find evidence for some type of receptor-mediated

Many of the
behavioral effects of THC are similar to those produced by benzodiazepines,
barbiturates, muscimol, and alcohol.Namely, each of these has antianxiety, euphorogenic, muscle relaxant,
sedative-hypnotic, analgetic, cardiovascular, thermoregulatory, and memory
altering properties.It is therefore
relevant that all of the above compound classes act on the chloride channel
regulated by the gamma aminobutyric acid (GABA) type A receptor complex to
increase inhibitory chloride influx, and also interact synergistically with THC
in the production of certain behaviors (Olsen, 1982; Harris and Allan, 1989;
Sanna et al., 1990; Pertwee et al., 1988).

However, we
and others have observed that THC does not compete directly for the GABA, the
BZ, or the barbiturate-picrotoxin-hypnotic steroid receptors of this complex
(Koe et al., 1985; Majewska et al., 1986; Lawrence et al., 1985).For example, we found that 1 mM
(-)-delta-9-THC displaced neither [3H]muscimol, [3H]flunitrazepam, nor
[3H]t-butylbicycloorthobenzoate (TBOB) (B.E. Morton et al., unpublished).This suggests either that additional
unrecognized receptors exist as part of the GABA-A receptor complex, or that
some form of cascade coupling exists between the cannabinoid receptor and the
chloride channel.Thus, it was of
interest that the regional distribution of the newly discovered cannabinoid
receptor was remarkably similar to that of the GABA-A receptor complex
(Herkenham et al., 1991a; de Blas et al., 1988; Herkenham et al., 1991b).

Here, we
report that THC causes an increase in GABA-muscimol mediated 36-chloride uptake
into brain synaptoneurosomes.The effects
of THC to increase chloride uptake were blocked by the GABA-A chloride channel
antagonists, bicuculline and picrotoxin.These results suggest that THC in some way increases chloride
influx-mediated

inhibitory hyperpolarization.

2.MATERIALS AND
METHODS

In brief,
synaptoneurosomes were prepared from whole rat brain, and the effects of
several cannabinoid compounds and other drugs upon synaptosomal 36-chloride
(36Cl) uptake was assayed using slightly modified versions of standard test
tube and filtration procedures (Harris and Allan, 1985; McQuilkin and Harris,
1990).

Preparation
of Synaptoneurosomes (SNs):The method
of Harris (Harris and Allan, 1985; McQuilkin and Harris, 1990) was adapted for
use with rats.Fresh (<1 hr) rat
brain was washed with buffer and homogenized in 5 ml assay buffer with 10 up
and down strokes of a glass-teflon homogenizer.The resulting crude homogenate was placed in a 15 ml polystyrene
tube and centrifuged for 15 minutes at 1000 x g (SS34 rotor).The supernatant fraction was discarded and
the pellet was gently resuspended in 10 ml assay buffer with a glass-teflon
homogenizer.Assay buffer was 145 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM D-glucose, 1 mM CaCl2, and 10 mM HEPES,
adjusted to pH 7.4 with Tris base.The
suspension was placed in a 15 ml polystyrene tube and vortexed, followed by
centrifugation (15 minutes, 1000 x g).The pellet was resuspended with a glass-teflon homogenizer in enough
buffer to create 10 ml total volume.100 ul of this was assayed for protein content.After assaying the amount of protein
present, enough buffer was added to make a final SNs preparation of 2.5 mg/ml
protein.Properties of similar
osmotically sensitive vesicular preparations from guinea pig brain have
described (Hollingsworth et al. 1985).

Synaptoneurosome Preincubation:To each of one set of tubes, 300 ul SNs were added.Another set of tubes contained 36-Cl and
various drugs.All tubes were
preincubated in a 34 degree C water bath for 5 minutes, and then incubated in
the same bath as below.An alternative
approach was that THC was sometimes added to SNs just before placement in the
water bath.This was done in order to
see the effect of THC preincubation on chloride channel function.

The
hydrophobicity of THC prevents its easy solubilization in aqueous media
(Garrett and Hunt, 1974).Use of PVP-40
promoted dispersal of THC, creating an opaque emulsion which could be used in
the examination of THC's effect on chloride influx in synaptoneurosomes.To avoid the effects of rapid cannabinoid
decomposition, fresh THC solutions were utilized.With these precautions, we were able to observe the effect of THC
on chloride influx.THC, prepared in a
colloidal suspension with 3%, w/v, polyvinylpyrrolidone-40 (PVP40), was active
in our hands over the concentration range of 100-1000 uM.

Synaptoneurosome Incubation-Separation Methods:The following two methods were used
interchangeably:Test tube method:A tube with the 36-Cl/drug and a tube with
synaptoneurosomes (SNs) were removed from the 34 degrees C water bath.The tube with SNs was vortexed and 200 ul of
the SNs was removed.The tube with the
36-Cl/drug was then vortexed, and while vortexing, the 200 ul of the SNs was
rapidly delivered into the drug tube.After 3 seconds, over which time chloride uptake has been shown to be
linear (Harris and Allan, 1985), 4 ml of ice cold 100 uM picrotoxin/buffer was
rapidly added into the drug tube, to quench the influx.Tube contents were poured rapidly into a presoaked
GB100R filter (Fisher Scientific) on a Hoefer FH224V filtration unit (manifold
on) at a

second each, of 4 ml 100 uM picrotoxin/buffer were added
to the filter (manifold off).

Filter
Method: 1 ml of preincubated SNs (0.5 mg protein/ml) were poured on a filter
with the manifold valve of the filtration unit opened and manifold tower
removed.The valve was closed and 1 ml
of 36-Cl/drug added to the filter.After 3 seconds, the manifold valve was reopened and 3 ml assay buffer
containing ice cold 100 uM picrotoxin was poured upon the filter, which was
then washed with 8 ml ice-cold assay buffer containing 100 uM picrotoxin.

Scintillation Counting:After
washing, filters were placed in glass scintillation vials.5 ml of ScintiVerse scintillation fluid
(Fisher Scientific) was added to each vial.Vial radioactivity was then measured with a scintillation counter.All methods used here sought to be identical
or equivalent to those developed in R. A. Harris' laboratory (Harris and Allan,
1985; McQuilkin and Harris, 1990).

Statistical
Treatment of Data:Significant 36-Cl
uptake differences between control synaptosomal samples and drug treated
samples were confirmed by computer assisted analysis of variance.

3.RESULTS

Using the
test tube 36-Cl assay, 200 uM THC enhanced 36-Cl uptake over that of the

reagent blank by an average of 20% (Figure
1).When the filter assay was used,
200 uM THC increased the uptake an average of 42% above control (Figure
1).Analysis of variance (ANOVA)
confirmed both of these increases to be highly significant (p=<0.05).

When the
effects of GABA on basal 36-Cl uptake were examined, half maximal stimulation
was found at 15 uM GABA with a maximum being reached by 100 uM at which point
uptake was at least doubled over that occurring in the absence of exogenous
GABA (Figure
3).In the presence of 1000 uM THC,
there was a leftward shift of GABA stimulation so that uptake was enhanced by
at least 20% for both 3 (p=<0.05) and 10 uM GABA (p=<0.05), falling off
at 30 uM GABA (p=<0.05), so that at the 100 uM maxima, no significant
increase was observed (Figure
3).Similar results were observed
repeatedly with muscimol (data not shown).

DISCUSSION

The
elevation of basal chloride uptake in synaptoneurosomes by THC was highly

reproducible.Cannabinol or cannabidiol (1 mM) were without effect, while 1 mM (+)-THC
was somewhat active, as had been reported in the case of the cloned cannabinoid
receptor (Matsuda et al., 1990). Since the THC-induced elevation in 36-chloride
uptake was blocked by picrotoxin, it demonstrates that the flux of chloride
through a chloride channel was affected by THC (Lawrence et al., 1985; Lawrence
and Cassida, 1983).Since bicuculline
also blocked the THC-induced increase in chloride uptake, this indicates that
the GABA-A receptor complex is involved (Mohler and Okada, 1978; Olsen and
Snowman, 1983).Although the Ki of THC at the cannabinoid receptor is
about 0.5 uM (Devane et al., 1988), THC was not active in less than micromolar
concentrations here.Difficulties in
solubilizing it, preventing its degradation, and avoiding its adsorption to
containers make it probable that the absolute THC concentration present in our
assays was significantly lower than calculated.

The increase
in 36-chloride uptake by THC, suggests three mechanistic interpretations:1. THC mobilizes some of the large
endogenous GABA pool to increase chloride influx indirectly.2. THC binds to a presently unidentified
allosteric activator site on the GABA-regulated chloride channel to cause an
increase in GABA affinity, as occurs in the case of benzodiazepine and
barbiturate-steriod binding to this complex.3. THC binds to the cannabinoid receptor, thereby inhibiting cyclic AMP
synthesis by adenylate synthetase via a Gi transducer (Howlett, Qualy, and
Katachatrian, 1986).Lowered levels of
cyclic AMP then reduce protein kinase A activity to phosphorylate, the chloride
channel of the GABA-A complex.In the
absence of phosphorylation which narrows this channel, chloride influx is
enhance (Browning et al., 1990; Porter et al., 1990).Although, each of these mechanisms would result in chloride uptake,
hyperpolarization, and neuronal inhibition to produce the GABA-A-like
intoxication symptoms of THC, the mechanism involving the cannabinoid receptor
is at present most attractive.

As this work
was nearing completion, an abstract appeared which described an increase of
whole-cell ion current in cultured rat hippocampal neurons that was produced by
THCwhich was mimicked by GABA addition
(Hampson et al. 1990).A preliminary
report ofour own work has also
appeared (Shirae and Morton, 1991).

ACKNOWLEDGEMENTS

We thank R.
A. Harris for assistance in setting up the synaptoneurosome chloride uptake
assay, The Department of Biochemistry and Biophysics for some supplies and a
graduate stipend (to D.T.S.), and W. K. Ogata for technical aid on statistics
and graphics.

Legend:Circular data points depict the effects of
GABA alone.Triangular data points show
the effects of GABA in the presence of 1000 uM THC.*p=<0.05.3%PVP-40 was
present in all tubes.Other conditions
were as described in the test tube assay in the Figure 1 legend.